Metal coating useful for rendering the surface of the metal biocompatible

ABSTRACT

Compounds of formula (I), in which the groups R are hydrogen or C 1-4  alkyl, n is from 2 to 4, X is alkylene, poly(ethoxy) or an aryl-containing group, Y is a valence bond or a divalent functional or heterocyclic group or a trivalent alkylene group, and Z is a sulphur-containing group which contains a thiol or disulphide group are useful to provide biocompatible treatments of metal surfaces, such as silver and gold surfaces. Processes for the preparation of the compounds, intermediates useful in such processes, articles having a metal surface treated with such compounds and processes of rendering metal surfaces biocompatible which comprise treating the metal surfaces with them.

This application was filed under 35 U.S.C. 371 and was upon PCTInternational Application No. PCT/GB93/00853, filed Apr. 23, 1993.

This invention relates to new compounds useful as metal coatings torender metal surfaces biocompatible, a process for their preparation,and their use in rendering a metal surface biocompatible.

The clinical use of blood contacting devices and prostheses is of majorimportance today in cardiovascular surgery and other fields of medicine.Heart valves and blood vessel prostheses, balloon pumps and cathetersare being implanted in daily surgical practice to restore or diagnosecardiovascular function. Artificial organs are routinely employed inblood detoxification by absorptive haemoperfusion and in oxygenation(membrane oxygenators and heart-lung devices). Considerable effort andcapital is invested in Europe and the U.S.A. in the development andexperimental evolution of an implantable artificial heart system. Thedevices are commonly constructed from metals and, when in use, ablood-metal contact is present. This contact will cause a reaction inthe recirculating blood, which, depending on the choice of metal, thedesign parameters, the flow or the addition of the anticoagulants, maylead to protein deposition, adhesion and destruction of red blood cells(haemolysis), platelet (thrombocyte) adhesion and aggregation and bloodcoagulation leading to formation of a haemostatic plug (thrombus). Theoccurrence of thromboembolism in cardiovascular surgery continues to bea problem, notwithstanding routine treatment with anticoagulants. Forthese reasons the search for biocompatible non-thrombogenic materialshas been an important research objective over the last two decades.

In addition, certain diagnostic procedures designed for the rapidanalysis of analytes in body fluids are compromised by non-specificbinding of fluid components. This problem is particularly acute inbiosensors which use parameters such as the mass or the refractive indexof the analyte to determine analyte concentration. For instance, smallchanges in refractive index associated with the binding of body fluidcomponents to a metal film can be measured using surface plasmonresonance (SPR).

We have now devised new compounds which aim to mimic some of theinterfacial characteristics of the outer cell surface of for example redblood cells, and in particular the lipid component of the biologicalmembrane which is the simplest common factor of such surfaces. Thecompounds are derivatives or analogues of phosphorylcholine that can belinked to the metal surface which is to be rendered biocompatible via athiol or disulphide functional group in the compounds. This deposits aphosphorylcholine type of residue on a surface. Such residues arecommonly found in lipid membranes.

Accordingly the present invention provides compounds of formula (I)##STR1## in which the groups R are the same or different and each ishydrogen or a straight or branched C₁ -C₄ alkyl group, preferablymethyl; n is from 2 to 4, preferably 2, and X is a straight or branchedC₁₋₃₀ alkylene group, preferably a group of formula --(CH₂)_(a) --, or Xis a group of formula --(CH₂ CH₂ O)_(b) --, or --(CH₂)_(c)--Ar--(CH₂)_(d) -- where a is from 1 to 30, b is from 1 to 20, c and dare the same or different and each is from 0 to 5, and Ar is a para- ormeta-disubstituted aryl group such as a phenyl, biphenyl or naphthylgroup (preferably a para-disubstituted biphenyl group) which isoptionally further substituted by one or more C₁ -C₄ alkyl groups; andeither

Y is a valence bond or a divalent functional or heterocyclic group; and

Z is hydrogen or a group --SZ¹ where Z¹ is an alkyl, cycloalkyl,alkylcycloalkyl, aryl, alkylaryl, heterocyclic, alkylheterocyclic groupor a group of formula (II): ##STR2## where Y, X, R and n are ashereinbefore defined; or Y is a trivalent alkylene group,

Z is a group --SZ¹ and

Z¹ is an alkylene group, unsubstituted or substituted by alkyl, aryl,alkylaryl, cycloalkyl or alkylcycloalkyl groups and bonded to the groupY so --Y--S--Z¹ form a 5 to 8 membered, preferably 5 or 6 membered, ringcontaining a disulphide linkage;

or a hydrate thereof.

Without wishing to be limited by the theory of the invention, it isthought that when metal surfaces are treated with the compounds of thepresent invention, the thiol or disulphide bond, is cleaved and a newbond is formed between the metal surface and the sulphur atom.

Particularly preferred compounds of formula (I) are those in which X is--(CH₂)_(a) -- and a is from 1 to 30, preferably 1 to 20, morepreferably 12 to 18. In an alternative embodiment x is a group--(CH₂)_(a) -- where a is from 1 to 8, more preferably 2 to 6. Otherpreferred compounds are those wherein X is --(CH₂ CH₂ O)_(b) -- and b isfrom 1 to 7: those compounds in which X is --(CH₂ CH₂ O)_(b) --,particularly when b is higher than 7 (e.g. 8 to 10), tend to exist asmixtures of compounds with different values of b rather than as puresingle compounds. The value of b may, therefore be fractional,representing an average value for the mixture of these compounds. Thecompounds in which X is --CH₂ (p--C₆ H₄) CH₂ --, --CH₂ (p--C₆ H₄)--,--(p--C₆ H₄) CH₂ --, --(p--C₆ H₄)--, --CH₂ (p--C₆ H₄ C₆ H₄)CH₂ --, --CH₂(p--C₆ H₄ C₆ H₄)--, --(p--C₆ H₄ C₆ H₄)CH₂ -- or --(p--C₆ H₄ C₆ H₄)-- arepreferred.

Compounds of formula (I) in which R is hydrogen, methyl, ethyl, n-propylor n-butyl are also preferred, as are compounds in which all the Rgroups are the same.

Particularly preferred are the compounds of formula (I) which contain aphosphorylcholine moiety, ie in which each R is methyl and n is 2.

One particularly preferred combination is when the compound of formula(I), contains a phosphorylcholine moiety and X is a group of formula--(CH₂)₁₂ -- or --(CH₂)₆ --, i.e. dodecoxy- orhexoxyphosphinyloxy-N,N,N,-trimethylethanaminium hydroxide inner salts.

As the divalent functional or heterocyclic group, Y, mention may be madein particular of the following combination of S-Y:

--S(X¹)C(═T)N(H)--, where X¹ is as defined above, preferably a straightor branched chain alkylene group, containing from 1 to 20 carbon atoms,such as (CH₂)₁₋₂₀, preferably (CH₂)₁₋₆, e.g. (CH₂)₂, and T is oxygen orsulphur, preferably oxygen;

--SC(═T)-- where T is oxygen, sulphur or NH, preferably oxygen orsulphur;

--SC(═T)N(H)-- where T is oxygen, sulphur or NH, preferably oxygen orsulphur; or

--SHet-Y¹ -- where Y¹ is a single bond, oxygen or sulphur, or an alkoxyor alkylthio group containing from 1 to 10 carbon atoms, and Het is aheterocyclic group, e.g. a pyridyl, pyrazinyl, pyriminidyl, triazinyl,quinolyl, isoquinolyl, pyrrolyl, furyl, thienyl, thiazolyl,isothiazolyl, diazathiazolyl, e.g. 1,3,4 thiadiazolyl, piperidyl,piperazyl and sugar rings, e.g. glucose. Particular mention may be madeof the following linking groups containing heterocyclic rings: ##STR3##

If the group Z¹ is an alkyl group it may be straight or branched andcontain typically from 1 to 10 carbon atoms.

If Z¹ is a cycloalkyl or alkyl cycloalkyl group then typically thecycloalkyl ring contains from 5 to 8 carbon atoms and is unsubstitutedor substituted by one or more alkyl groups, typically containing from 1to 4 carbon atoms. In the case of an alkylcycloalkyl group the alkylportion typically contains 1 to 10 e.g. 1 to 6 carbon atoms and isstraight or branched.

If Z¹ is an aryl or alkylaryl group, then typically the aryl is a phenylor naphthyl ring which is unsubstituted or substituted by one or morealkyl groups, typically containing from 1 to 4 carbon atoms. In the caseof an alkylarylalkyl group the alkyl portion typically contains 1 to 10e.g. 1 to 6 carbon atoms and is straight or branched.

If Z¹ is a heterocyclic or alkyl heterocyclic group the typically theheterocycle is a pyridyl, pyrazinyl, pyriminidyl, triazinyl, quinolyl,isoquinolyl, pyrrolyl, furyl, thienyl, thiazolyl, diazathiazolyl, e.g.1,3,4-diazathiazolyl, piperidinyl or piperazyl group, which isunsubstituted or substituted by one or more alkyl groups containingtypically from one to four carbon atoms. A particularly preferredembodiment is when Z¹ is a 2-pyridyl group.

If the group Z¹ is a group of formula (II) then it is preferred that thecompound of formula (I) is a symmetrical disulphide.

Where the group Z¹ is an alkylene group bonded to the trivalent alkylenegroup Y to form a ring containing a disulphide linkage, then preferablyZ¹ --S--S--Y-- is a group of formula ##STR4## wherein K and K' are thesame or different and each is a valence bond or an alkylene group of 1to 5 carbon atoms, unsubstituted or substituted by alkyl, aryl,alkylaryl, cycloalkyl or alkylcycloalkyl groups, provided that the ringcontaining K and K' is a 5 to 8 membered ring, preferably 5 to 6membered, ring.

Preferably, where K or K' is an alkylene group it is unsubstituted, orif substituted, the substituents are alkyl, aryl, alkylaryl, cycloalkylor alkylcycloalkyl groups as described above in relation to Z¹. It ispreferred that K be a group --(CH₂)₂₋₅ --, e.g. --(CH₂)₂ --, and K'valence bond.

According to a further feature of the present invention, there isprovided a process for preparing the compounds of formula (I) whichcomprises:

(a) reacting a compound of formula (III)

    Z.sup.1 --S--S--Y--(X)--OH

in which X and Z¹ are as hereinbefore defined and Y, with a compound ofthe formula (IV) ##STR5## in which n is as hereinbefore defined and Halis a halogen, preferably chlorine, to provide a compound of formula (V)##STR6## in which Z¹, X and n are as hereinbefore defined, reacting thecompound of formula (V) with NR₃, where R is as hereinbefore defined toprovide a compound of formula (I) wherein Z is a group Z¹ S, and Y is avalence bond or a trivalent alkylene group bonded to Z¹ to form a ringcontaining a disulphide linkage;

(b) converting a compound of formula (VI) ##STR7## where Q is a halogen,preferably chlorine, bromine or iodine, e.g. bromine, or Q is a readilydisplaceable leaving group, such as tosyl or mesyl, or Q is a protectedthiol group, e.g. a thioether or thioester, Y is a valence bond or adivalent heterocyclic group and X and R and n are as hereinbeforedefined, to a compound of formula (I) in which Z is hydrogen, and ifdesired converting the compound thus obtained to a disulphide of formula(I) in which Z¹ is a group of formula (II);

(c) reacting a compound of formula (VII) ##STR8## where Q¹ is halogen,e.g. chlorine or bromine or a readily displaceable leaving group, suchas tosyl or mesyl, T is oxygen or sulphur, and X, R and n are ashereinbefore defined with a sulphur-containing compound e.g. sodiumsulphydride, to form a compound of formula (I) where Y is a group offormula, >C═0 or >C═S and Z is H

(d) reacting a compound of formula (VII) where T is sulphur with analcohol and then ammonia to provide a compound of formula (I), where Yis a group of formula >C═NH and Z is H;

(e) reacting a compound of formula (VIII): ##STR9## where X, R and n areas defined hereinbefore with a compound of formula (IX). ##STR10## whereZ¹ and X are as hereinbefore defined, T is oxygen or sulphur and Q³ is areadily displaceable group, such as halogen, oxyamino, e.g.N-succinimidyl or a group of formula ##STR11## (Z¹ and T being the sameas in formula (IX)), to form a compound of formula (I) in which Z is S,and Y is --C(═T)NH--;

(f) reacting a compound of formula (X) ##STR12## where T is oxygen orsulphur and Q¹, X, R and n are as hereinbefore defined with a sulphurcontaining compound e.g. sodium sulphydride to fork a compound offormula (I) where Y is a group of formula --C(═T)NH-- and Z is H;

(g) reacting a compound of formula (X) as hereinbefore defined where Tis sulphur with an alcohol and then NH₃ to provide a compound of formula(I) where Y is a group of formula --C(═NH)NH-- and Z is H;

(h) converting a compound of formula (I) where Z is hydrogen to acompound of formula (I) where Z is a group SZ¹, as hereinbefore defined;or

(i) converting a compound of formula (I) where Z is a group SZ¹, ashereinbefore defined to a compound of formula (I) where Z is hydrogen;and if desired, converting the resulting product to a hydrate thereof.

The invention also provides, as a further feature, the new compounds offormula (VI), (VII) and (X) as hereinbefore defined, which are useful asintermediates in the preparation of compounds of formula (I).

For the preparation of a compound of formula (I) by route (a), thealcohol (III) is typically dissolved in an organic aprotic solvent(typically acetonitrile, N,N'-dimethylformamide, or dichloromethane forexample acetonitrile) and then treated with one equivalent of a compoundof formula (IV), e.g. 2-chloro-2-oxo-1,3,2-dioxaphospholane, in thepresence of a base (typically sodium or potassium carbonate,triethylamine or N,N,dimethylaminopyridine for example anhydrous sodiumcarbonate). This gives a cyclic phospholane of formula (V) as anintermediate which is typically treated with appropriate nitrogenousbase (for example trimethylamine) under anhydrous conditions in apressure bottle with an aprotic solvent (for example acetonitrile). Thisreaction is generally performed for 3 to 73 hours, (typically 18-24hours, for example 18 hours) at a temperature of 0 to 100° C.,(typically 60° to 75° C. for example 70° C.). The resulting compound offormula (I) may be isolated by column chromatography, using for examplesilica gel, or by for example crystallisation.

The compounds of formula (III) in which Y is a valence bond may beobtained by reacting a hydroxythiol compound of formula (XI)

    HS--(X)--OH                                                (XI)

where X is as hereinbefore defined with a disulphide (Z¹ S)₂, generallyin an organic solvent (for example ethanol and acetic acid mixtures) andisolated by silica gel chromatography. Typically the reaction isperformed at 0°-40° C. for example for 18 hours. The hydroxythiols offormula (XI) may be obtained commercially or by known method.

The compounds of formula (III) in which Y is bonded to Z¹ to form a ringcontaining a disulphide linkage may be obtained by intra-molecularoxidation of the corresponding dithiol to form a disulphide using forexample hydrogen peroxide, see for example Vogel, Practical OrganicChemistry, by B. S. Furniss, A. J. Hannaford, P. W. G. Smith and A. R.Tatcheil, Published by Longman, 1989. The corresponding dithiolcompounds may be obtained from dibromo compounds by reaction with asulphur containing compound, e.g, sodium sulphydride, thiourea or sodiumthiosulphate, or these compounds may be commercially available. Thedibromo compounds may be obtained commercially or using knowntechniques.

Where it is desired to prepare a compound of formula (I) by method (b),when Q is halogen or a readily displaceable group such as tosyl or mesylthen an appropriate compound of formula (VI) may be converted to acompound of formula (I), where Z is hydrogen by reaction with variousreagents (typically thiosulphate, e.g. sodium or potassium thiosulphate,then hydrochloric acid, sulphydride e.g. sodium sulphydride or thioureaand sodium or potassium hydroxide). The product thus obtained may bepurified, for instance by chromatography (typically silica gel, oralumina) and/or isolated for instance by crystallisation (in for examplemethanol and acetone). Where it is desired to prepare a compound offormula (I) by method (b), when Q is a protected thiol group thensuitable protecting groups include thioethers, e.g. tritylthioether,tert-butylthioether, 2-4-dinitrophenylthioether and benzylthioether,thioesters and silylthioethers e.g. diphenylmethylsilylether. Thesegroups may be added and removed using known techniques as described forexample in Vogel, supra, and Advanced Organic Chemistry, J. March,published J. Wiley, 3rd edition 1985.

If for example a thioether such as tritylthioether is used then a rangeof conditions (typically trifluoroacetic acid, in methanolic hydrogenchloride, hydrobomic acid in acetic acid or for example silver nitratein methanol) at temperatures of 0° to 100° C. (for example 30° C.) maybe used to convert an appropriate compound of formula (IV) to a compoundof formula I in which Z is hydrogen. The product may be purified bychromatography (for example silica gel) or by crystallisation (forexample from methanol or acetone).

The compounds of formula (VI) may be obtained by a procedure analogousto that described under (a), but starting from compounds of formula(XII)

    Q--(X)--OH                                                 (XII)

The compounds of formula (XII) may be obtained commercially or by usingknown methods. In the case where Q is --SCPh₃, they may in particular beprepared by the reaction of mercaptotriphenylmethane with an inorganicbase (for example potassium carbonate) followed by reaction with acompound of formula (XII) in which Q is halogen, e.g. bromine, or tosylor mesyl in an aqueous solvent mixture (for example water and ethanol).

The compound for formula (I) in which Z is hydrogen, thus produced, maybe converted to a disulphide in which. Z is Z¹ S and Z¹ is a group offormula (II) using known methods for the formation of disulphides formthiols, using for example hydrogen peroxide as described in Vogel,supra.

If it is desired to obtain a compound of formula (I) by route (c) or (d)the appropriate compound of formula (VI) may be first converted to ametallo reagent e.g. a Grignard reagent by reaction with magnesium byknown methods. The metallo-derivative may then be reacted with acompound Q¹ ₂ C═T where each Q¹ is the same or different and is asdefined above, typically in the presence of a base e.g. triethylamineand Li₂ CuCl₄.

According to (c) a compound of formula (VII) which may be obtained thusmay be treated with a sulphur containing compound, e.g. sulphydride,thiosulphate or thiourea, e.g. sodium sulphydride to obtain a compoundof formula (I) where Y is >C═O or >C═S.

According to (d) a compound of formula (VII) in which T is sulphur maybe treated with an alcohol, e.g. ethanol, followed by ammonia to obtaina compound of formula (I) where Y is >C═NH.

If it is desired to prepare a compound of formula (I) by route (e) anappropriate compound of formula (VIII) is reacted with an appropriatecompound of formula (IX). Typically the compound of formula (VIII) istaken up in a solvent, typically an aqueous buffer or a polar aproticorganic solvent (for example dimethyl sulphoxide). The compound offormula (IX) is then added in an organic solvent, typically ethanol or amixture of dimethylsulphoxide and triethylamine. The mixture isgenerally stirred at 0° to 40° C. (for example 27° C.) for one to 24hours (for example 18 hours) and the product typically purified bycolumn chromatography.

The compounds of formula (IX) may be prepared by known methods. Forexample compounds of formula (IX) may be obtained by reaction of a thiolHS--X--C(═T)Q³ with a disulphide (Z₁ S)₂. This reaction may be performedusing the conditions described for the formation of the disulphides offormulae (III) described above. The thiols HS--X--C(═T)Q³ may beprepared using known techniques.

The compounds of formula (VIII) may be prepared by a procedure analogousto that described above in relation to (a), but starting from aprotected amino alcohol of formula (XII):

    R'R"N--(X)--OH                                             (XIII)

The coupling of the N-protected alcohols of formula (XIII) to thecompounds of formula (IV) may be performed in the presence of a baseunder anhydrous conditions. The reaction is typically performed at atemperature from -5° to 50° C. (preferably 10° to 30° C., e.g. 25° C.)in a dry organic solvent, e.g. acetonitrile or N,N-dimethylformamide andin the presence of an organic base, such as a tertiary amine, e.g.triethylamine or pyridine, or an inorganic base, such as an alkali metalcarbonate, e.g. sodium carbonate.

The ring opening reaction may, for example, be performed in tertiaryamine, e.g. trimethylamine, at a temperature from 20° to 100° C.,preferably 40° to 80° C., e.g. 70° C., and in a sealed pressure vesselfor 3 to 72 hours (e.g. 18 hours).

The deprotection may be performed as a separate step after or, in somecases, before the ring-opening reaction. It may also be performed at thesame time as the ring-opening reaction.

The protecting groups are chosen so that they do not react with thecompounds of formula (IV). As examples of particular protecting groupsthere may be mentioned:

amides (NR' and/or NR" is an amide group), e.g. N-phthalimides;

carbamates (NR' and/or NR" is a carbamate group), e.g.9-fluorenylmethoxycarbonylamines, or teftbutyloxycarbonylamines;

hindered secondary amines, (R' is a hindered group e.g. triphenylmethyland R" is H); or salts, (NR'R" is a NH₃ ⁺ A⁻ group). Suitable counterions A⁻ are anions of organic acids, such as acetic or p-toluenesulphonic acid or inorganic acids such as hydrogen halides, e.g.hydrogen chloride.

The N-protected aminoalcohols of formula (XIII), may be prepared frombromoalcohols of formula (XIV) or aminoalcohols of formula (XV) whichare commercially available or may be prepared by known methods:

    Br--(X)--OH                                                (XIV)

    H.sub.2 N--(X)--OH                                         (XV)

In some cases however, the protected amine alcohols are themselvescommercially available e.g. N-(2hydroxyethyl)phthalimide.

In the case where the protecting group is an amide the protected aminoalcohol may be prepared from either the bromoalcohol of formula (XIV) orthe aminoalcohol of formula (XV) by known methods. For example if theprotecting group is a phthalimide, the protected amino alcohol isobtained by reaction with an alkali metal phthalimide, e.g. potassiumphthalimide. Typically the reaction with phthalimide is performed in anorganic solvent such as N,N-dimethylformamide at a temperature from 70°to 110° C. e.g. 90° C. After coupling to a phosphorus compound offormula (IV) and ping-opening, deprotection is performed under basicconditions (for example, in aqueous hydrazine). This gives the finalproduct of formula (VIII) which can be purified for instance by columnchromatography using, for example, silica gel.

In the case where the protecting group is a carbamate, protection isafforded by reaction of an amino alcohol with, for example, achloroformate or acid anhydride to give a carbamate. The reaction isgenerally performed in an organic solvent, at a temperature from 10 to50° C. and in the presence of a base. 9-Fluorenylmethoxychloroformate,for example, reacts with amines to give 9-fluorenylmethoxycarbonylaminederivatives and di-tert-butyldicarbonate reacts with amines to givetert-butyloxycarbonylamine derivatives. Ethanolamine, for example,reacts with 9-fluorenylmethoxychloroformate under anhydrous conditionsin an inert solvent such as dichloromethane, in the presence of asuitable base such as pyridine, in a temperature range of, for example,-10° C. to 50° C., for example, 10° C., to giveN-9-fluorenylmethoxycarbonylaminoethanol. Ethanolamine reacts withdi-tert-butyldicarbonate under aqueous conditions, for example, aqueous1,4-dioxan, in the presence of a suitable base, for example sodiumhydroxide, at a suitable temperature, for example -10° C. to 50° C.,preferably at 0° C., to give N-tert-butyloxycarbonyl-aminoethanol.

The carbamate protecting groups may be removed after the couplingreaction by known methods. For example the N-9-fluorenylmethoxycarbonylamine protecting group may be removed under basic conditions in asuitable solvent, such as acetonitrile. Suitable bases for aminedeprotection include ammonia, dialkylamines such as diethylamine,trialkylamines such as trimethylamine, cyclic amines and especiallycyclic secondary amines such as morpholine, piperazine, piperidine anddiazabicyclic bases such as 1,5-diazabicyclo(4.3.0)non-5-ene (DBN) and1,8-diazabicyclo(5.4.0)undec-7-ene (DBU). The deprotection conditionsmay be chosen such that deprotection is performed prior to ring-openingor at the same time. The tert-butyloxycarbonyl amine protecting groupmay be removed using a suitable acid, for example trifluoroacetic acidor hydrochloric acid. The reaction may be performed in a suitablesolvent system, for example, 1,4-dioxan/chloroform mixtures at atemperature of 0° to 50° C., for example, 21° C.

In the case where the protecting group is a hindered secondary amine theprotected aminoalcohol (VIII) may be prepared by initial blocking of thehydroxyl function (for example, by reacting with chlorotrimethylsilane)in an organic solvent (for example, tetrahydrofuran) in the presence ofan organic base (for example triethylamine). The amine function is thenprotected using a hindered chloroalkane (for example,chlorotriphenylmethane) in the presence of an organic base (for example,triethylamine). The hydroxyl function is then deprotected under mildconditions (for example, with methanol).

After coupling and ring opening, deprotection may be performed underacidic conditions, for example, with trifluoroacetic acid or withhydrogen chloride gas, in a non-aqueous solvent, for example,1,4-dioxan, or chloroform. This gives the crude product which can bepurified by column chromatography using, for example, silica gel.

If NR'R" is NH₃.sup.⊕ A.sup.⊖ in the protected aminoalcohol of formula(VIII) , it will react with the compound of formula (VIII) selectivelyvia the hydroxyl group. Protected aminoalcohols in which NR'R" isNH₃.sup.⊕ A.sup.⊖ are prepared by protonation with a suitable acid.Suitable acids include inorganic and organic acids especiallyp-toluenesulphonic acid which gives with, for example, ethanolamine, acrystalline p-toluenesulphonate which is soluble in a solvent suitablefor the reaction with (III), for example, acetonitrile.

After coupling, these amine salts may be converted to free amines undersuitable basic conditions using, for example, trimethylamine.Advantageously, the protected amine salts are ring-opened and convertedto free amines in a single step using trimethylamine. In the case wherethe acid addition salt is desired it is not necessary to deprotect theamine group.

If it is desired to obtain a compound of formula (I) using method (f) or(g), the appropriate compound of formula (VIII) may be first reactedwith a compound, Q¹ ₂ C═T typically in the presence of a base to providethe appropriate compound of formula (X). According to (f) the compoundof formula (X) may then be reacted with a sulphur-containing compound,e.g, a sulphydride, thiosulphate or thiourea, e.g. sodium sulphydride toobtain a compound of formula (I) in which Y is --C(O)NH-- or --C(S)NH--.According to (g) the compound of formula (X) may be treated with analcohol, e.g. ethanol, followed by ammonia to obtain a compound offormula (I) in which Y is --C(═NH)NH--.

If it is desired to obtain a compound of formula (I) by route (h), thenan appropriate compound of formula (I) where Z is hydrogen, may bereacted with a disulphide of formula (Z¹ S)₂. Generally the reaction isperformed in an organic solvent (e.g. ethanol and acetic acid mixture).Typically the reaction is performed at 0°-40° C. for example for 18hours.

The compounds of formula (I) in which Z is Z¹ S may be converted tocompounds of formula (I) in which Z is hydrogen according to method (i)using known methods for the formation of disulphides. For example thereaction may be performed using zinc in dilute acid, e.g. HCl or usingtriphenylphosphine in water as described in March and Vogel, supra.

Hydrates of the compounds of formula (I) may be produced by the abovemethods or they may be formed by an additional separate step, usingknown methods.

As a further feature, the present invention provides a process forrendering a metal surface biocompatible, which process comprisesapplying to the surface a compound of formula (I) or a hydrate thereof.

Metals which may be derivatised in this way include aluminium, tin,titanium, iron, silver, gold, platinum, chromium, copper, nickel,palladium, tungsten and alloys containing these. In particular thecompounds of formula (I) may be used to treat silver and gold.

Treatment may typically be affected with a solution of the compound offormula (I) or hydrate thereof in for example aqueous buffer, e.g.phosphate buffer, methanol or ethanol. The treatment is typicallycarried out at a temperature from -20° to 100°, preferably 0° to 50° C.,e.g. about 27° C., and for period of typically up to 96 hours. Treatmentmay be carried out for example, in methanol, ethanol or aqueous buffer,e.g. phosphate buffer. If aqueous buffer is used the pH is typicallyfrom 4 to 9, such as 5 to 8, preferably about 7.5.

Pre-treatment of the metal surface, for example by heating, electrolysisor chemical activation may be required in order to enhance thereactivity of some metal surfaces towards the compounds of formula (I).

Silver may for example be readily treated with a compound of formula (I)or a hydrate thereof at 0° to 50° C., e.g. ambient temperature, in anorganic solvent, for example methanol, or in phosphate buffer, typicallyat a pH from 4 to 9, preferably 5 to 8, e.g. 7.5, for 24 hours. Gold maybe treated at typically 0° to 50° C., e.g. ambient temperatures with asolution of compound formula (I) or a hydrate thereof 1 to 96 hours.Other metals, such as titanium, platinum, chromium and copper may beheated (for example at 50° to 250° C.) prior to exposure to a compoundof formula (I), which is for example a thiol, or a hydrate thereof.Other metals e.g. palladium and platinum may be reacted with a compoundof formula (I) or hydrate thereof under electrolytic conditions. Metalssuch as aluminium may require chemical activation before derivatisationsee for example Advanced Inorganic Chemistry, by F. A. Cotton and G.Wilkinson 3rd Edition 1972, published by Interscience.

As a further feature the invention also provides an article having ametal surface to be introduced into the human or animal body or which isto be brought into contact with body cells or fluids or into contactwith protein solutions which surface has been treated with a compound offormula (I) or a hydrate thereof.

Articles having metal surfaces to which compounds of formula (I) havebeen attached show reduced protein adsorption at that surface andincreased haemocompatibility.

The present invention will now be further illustrated by means of thefollowing Examples:

EXAMPLES

The following assays have been used to evaluate coatings on surfaces ofcompounds according to the present invention.

Protein adsorption using an enzyme immunoassay

The assay determines absorption of human fibrinogen at a surface. Thisprotein is representative of protein which is typically adsorbed at asurface. The assay can be readily modified to determine the absorptionof other proteins.

Discs (5 mm in diameter) of metal (as controls) and metal treated withcompound as described below, were prepared and washed with phosphatebuffered saline (PBS) for at least 10 minutes in the wells ofmicroplates. The samples were incubated with human plasma (300 μl) for10 minutes and then washed with PBS three times. Each of the testsamples and each of the control samples were treated with humanfibrinogen-specific antibody (300 μl) for 30 minutes and again washedwith PBS three times. As a control for non-specific binding of antibodyto the samples, each sample was also incubated with non-specificantibody (300 μl) for 30 minutes. A conjugate of horseradish peroxidaseand a second antibody specific to the first antibody (300 μl) was addedto both the test samples and the controls and incubated for 30 minutesbefore washing. Each of the test samples and the controls weretransferred to new microplates and a solution of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) in phosphate-citrate buffer (300μl,0.6 mg/ml) added, the reaction was allowed to proceed for 10 minutes.At this time an aliquot of the mixture (200 μl) was removed and added toa solution of citric acid and sodium azide in distilled water (20 μl,0.21 g/ml and 2 mg/ml respectively). The optical density of thesolutions was measured using a Techgen automated plate reader at 650 nmusing the ABTS solution as blank.

In an alternative procedure, rather than using ABTS, each of the sampleswas transferred to wells of new microplates and a solution ofo-phenylene diamine (OPD) in phosphate-citrate buffer (300 μl, 0.4mg/ml) added, and the reaction was allowed to proceed for 10 minutes. Atthis time an aliquot of the mixture (200 μl) was removed from each welland the optical density of the solutions was measured using a Techgenautomated plate reader at 450 nm using the OPD solution as blank.

Activated Platelet Study

Blood was collected from a healthy adult volunteer using the doublesyringe method where the first 5 ml of blood is discarded. The blood wascollected into tri-Sodium citrate (32 g/1) in the proportion of 9volumes to 1 volume citrate in plastic tubes. The samples were kept atroom temperature on a spiral mixer until used.

Discs (5 mm in diameter) of metal as controls and material treated withcompounds as described below were prepared and placed into the wells ofa microplate. Half of the test replicates were incubated with citratedblood (200 μl) and the remainder were incubated with EDTA-treated bloodon a phase shaker for 30 minutes before washing in PBS four times.Platelet activation was measured by a propriety assay (EJ Campbell etal, Mat. Res. Soc. Symp. Proc. 252, 229-237). The procedure is analogousto that described above for detection of proteins by enzyme immunoassaybut uses antibodies against GMP140 to detect the presence of thisplatelet activation marker on the surface of biomaterials. In thepresence of EDTA, which extracts calcium from inside platelets,activation is inhibited, so that incubation with EDTA-treated blood actsas a non-specific control for activation, obviating the need forincubation in non-specific antibody.

Surface Plasmon Resonance

Surface plasmon resonance (SPR) is a biosensing technique which measuresminute changes in refractive index within a few hundred nanometres of athin metal film (Charles SA et al, Biotechnology & Human Predispositionto Genetic Disease, Symposia on Molecular & Cellular Biology,Wiley-Liss, 1990, vol 126, pp 219-228). For instance, sensitivemeasurements of the interaction of proteins with a metal surface can bemade in real time.

Silver films, 50 nm thick, were vacuum deposited onto 25 mm square glassmicroscope slides. The films were inserted into the SPR refractometer,and subjected to a flow (0.4 ml/min) of 1 μM human immunoglobulin G for10 minutes. The change in resonance angle was monitored continuously,and the total change compared to an untreated film.

EXAMPLE 12-[2{2'Pyridyldisulphide}ethoxyhydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminiumhydroxide inner salt ##STR13##

2,2'-Dipyridyl disulphide (15 g, 68 mmol) was dissolved in absoluteethanol (40 ml) and glacial acetic acid (1.4 ml). 2-Mercaptoethanol (3.3g, 42.5 mmol) in ethanol (20 ml) was added dropwise whilst stirring. Themixture was stirred for 16 hours at ambient temperature when thesolvents were removed under vacuum. The residue was treated with benzeneand evaporated under reduced pressure three times, and then dried undervacuum. The dried material was chromatographed on silica gel elutingwith n-hexane/diethylether mixtures. Fractions containing product wereevaporated to give the 2'-pyridyl disulphide -2-ethanol. ¹ H-NMR(CDCl₃), 60 MHz, 2.9 (t, 2×H), 3.8 (t, 2×H), 7.0-7.6(m, 3×H), 8.4 (d,1×H)ppm.

2-'Pyridyldisulphide-2-ethanol (5.3 g, 28 mmole) was stirred in dryacetonitrile (80 ml) together with anhydrous sodium carbonate (200 mg)under nitrogen for 90 minutes. Further2-chloro-2-oxo-l,3,2-dioxaphospholane (1 g, 7 mmol) was added andstirring maintained for 30 minutes. The mixture was filtered undernitrogen and carefully added to frozen trimethylamine (4.8 ml, 3.15 g,53 mmol) in a pressure tube which was sealed and heated at 70° C. for 16hours. The excess trimethylamine was removed, and the solvent evaporatedunder reduced pressure. The residue was chromotographed on silica gel,eluting with methanol. Fractions containing product were combined,evaporated and the residue triturated successively with acetone anddiethylether. Chloroform was added to the residue followed by acetoneuntil a pale gum was deposited. The solvents were decanted, the gumwashed with acetone and the residue dried under vacuum for three hoursto give2-[2{2'pyridyldisulphide}ethoxyhydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminiumhydroxide inner salt H¹ -NMR (300MHz) CD₃ OD: 3.10 (t, 2×H), 3.2 (s,9×H), 3.60 (m, 2×H), 4.1 (q, 2×H), 4.25 (m,2×H), 7.2 (t, 1×H), 7.6-7.9(m, 2×H), 8.4 (d,1×H)ppm.

Mass Spectrum, FAB, M⁺ =353.

EXAMPLE 22-[(6-Bromohexoxvhydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminium,inner salt ##STR14##

6-Bromohexan-1-ol (1 g, 5.5 mmol), 2-chloro-2-oxo-1,3,2-dioxaphospholane(0.78 g, 5.5 mmol) and anhydrous sodium carbonate (580 mg) were taken indry acetonitrile (50 ml) and stirred under nitrogen at ambienttemperature for 90 minutes. The mixture was filtered under nitrogen andthe filtrate evaporated to a smaller volume (ca 20 ml). The solution wasadded to frozen trimethylamine (0.5 ml, 5.5 mmol) in a pressure vesselwhich was sealed and heated at 60° C. for 96 hours. The mixture wasfiltered and the filtrate partitioned between chloroform (100 ml) andwater (100 ml). The aqueous layer was evaporated under reduced pressureand the residue chromatographed on silica gel, eluting with methanol.Fractions containing the compound were combined, evaporated underreduced pressure, treated with benzene and evaporated under reducedpressure and then dried under vacuum to give 2-[(6-bromohexoxy hydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminium, inner salt ¹ H-NMR (300MHz)CD₃ OD: 1.40-1.60 (m, 4×H), 1.60-1.75 (m, 2×H), 1.75-1.90 (m, 2×H), 3.12(s, 9×H), 3.3 (m, 2×H), 3.70 (q, 2×H), 3.8-4.1 (m, 4×H)ppm.

EXAMPLE 32-[6-Mercaptohexoxyhydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminium,inner salt

2-[(6-Bromohexoxyhydroxyphosphinyl)oxy]-N,N,N-trimethyl ethanaminium,inner salt (100 mg, 0.29 mmol) and a solution of sodium thiosulphate(0.1M, 300 μl) were combined. Methanol (5 ml) was added and the mixturewas heated at 80° C. for one hour. Further sodium thiosulphate (0.1M, 3μl) was added followed by hydrochloric acid (1.0M) until the pH reached1.0. The mixture was heated at 80° C. for 16 hours. The solvents wereevaporated under reduced pressure to leave2-[6-mercaptohexoxynydroxyphosphinyl)oxy]-N,N,N-trimethylethanaminium,inner salt as a gum containing inorganic salts.

EXAMPLE 4 2[N (3-(2-Pyridyldithio)propionyl)(6-aminohexoxvhydroxy-phosphinyl)oxy-N,N,N-trimethylethanaminiumhydroxide, inner salt ##STR15##

2-[(6-Aminohexoxyhydroxyphosphinyl)oxy-N,N,N-trimethylethanaminiumhydroxide, inner salt (90 mg, 0.28 mmol) was dissolved in dry dimethylsulphoxide (2 ml). Triethylamine (197 μl, 1.41 mmol) was added, followedby N-succinimidyl 3-(2-pyridyldithio)propionate (88 mg, 0.28 mmol). Themixture was stirred at ambient temperature for eighteen hours. Thesolvent was evaporated under vacuum at a temperature of 60° C. andre-evaporated from methanol. The residue was chromatographed on silicagel eluting with methanol. The relevant fractions were combined andevaporated to dryness to give 2[N(3-(2pyridyldithio)propionyl)(6-aminohexoxyhydroxy

inner phosphinyl)oxy-N,N,N-trimethylethanaminium hydroxide, salt

¹ H-NMR (300MHz) (CD₃ OD) 1.2-1.9 (m, 8×H), 2.6 (t, 2×H), 3.2 (m, 2×H),3.26 (s, 9×H), 3.6 (m, 2×H), 3.8 (m, 2×H) , 4.3 m,2×H), 7.2 (t, 1×H),7.8 (n, 2×H), 8.4 (d, 1×H)ppm.

EXAMPLE 5

A silver coated substrate was washed with ethanol and dried undervacuum. The substrate was placed in a solution of[2{2'-pyridyldithio}ethoxy-hydroxyphosphinyl)oxy]-N,N,N-trimethylethanimiumhydroxide, inner salt (164 mg, 0.46 mmol) in ammonium dihydrogenphosphate buffer (pH 7.5, 2 ml). The reaction mixture was left for 24hours at ambient temperature. The substrate was removed and successivelywashed with ammonium dihydrogen phosphate buffer (pH 7.5), water andmethanol. The substrate was dried under vacuum to give a substrate witha coating of phosphonyl choline derivative.

EXAMPLE 6(12-MercaptododecoxVhydroxyphosphinyl)oxy-N,N,N,-trimethylethaniniumhydroxide, inner salt ##STR16##

Trityl mercaptan (1.1 g, 4 mmoles) was dissolved in ethanol (60 ml) andwater (60 ml) and stirred under nitrogen. Potassium carbonate (0.7 g, 4mmole) was added and the mixture stirred at ambient temperature for 30minutes.

12-Bromododecan-1-ol (1 g, 4 mmole) was added and the mixture heated at80° C. for 16 hours. After cooling, a pink solution separated out, whichwas decanted. The residue was azeotroped with benzene to give12-tritylthiododecan-1-ol (1.53 g, 3.31 mmole, 83% yield).

12-Tritylthiododecan-1-ol (1.53 g, 3.31 mmole) was dissolved in dryacetonitrile (40 ml) and anhydrous sodium carbonate (80 mg) followed by2-chloro-2-oxo-1,3,2-dioxaphospholane (0.50 g, 3.5 mmole) inacetonitrile (20 ml) were added. The mixture was stirred under nitrogenfor two hours. The reaction mixture was filtered, added to an excess oftrimethylamine in acetonitrile and heated at 70° C. for 24 hours. Aftercooling, a yellow liquor was decanted from the mixture, and the residuewas chromatographed on silica gel, eluting with chloroform/methanol(1:1). Fractions containing product were combined, evaporated to drynessand azeotroped with benzene to give(12-tritylthiododecoxyhydroxyphosphinyl)oxy-N,N,N,-trimethyl-ethaniminium hydroxide, inner salt (0.51 g, 0.73mmole, 22% yield).

(12-Tritylthiododecoxyhydroxyphosphinyl) oxy-N,N,N-trimethylethaniminium hydroxide, inner salt (0.45 g 0.72 mmole), wasdissolved in methanol (10 ml) and hydrobromic acid in acetic acid (6.2ml) was added and stirred for ten minutes. Benzene was added and themixture azeotroped. The residue was triturated with ethyl acetate (×2)and acetone (×2), dissolved in methanol and reprecipitated with acetone.The solid was chromatographed on reverse-phase silica gel eluting withmethanol, and fractions containing product were concentrated to give animpure product, 383 mg. Final purification was achieved by a secondreverse-phase chromatography column, eluting with methanol: water (9:1),to give(12-mercaptododecoxyhydroxyphosphinyl)oxy-N,N,N,-trimethylethaniniumhydroxide, inner salt, 33 mg, 0.086 mmole, 12% yield.

¹ H-NMR (CD₃ OD), 200MHz, 1.34 (m, 16×H), 1.69 (m, 4×H), 2.74 (t, 2×H),3.29 (s, 9×H), 3.71 (m, 2×H), 3.94 (q, 2×H), 4.34 (m, 2×H)

EXAMPLE 7(2-mercaptoethoxyhydroxyphosphinyl)oxy-N,N,N,-trimethyletaniniumhydroxide, inner salt

The compound was prepared by a method analogous to that of Example 6,using 2-bromoethanol in place of 12-bromododecan-1-ol.

¹ H-NMR (CD₃ OD), 200MH_(z), 2.90 (t, 3×H), 3.25 (s, 9×H), 3.73 (m,2×H), 4.06 (q, 2×H), 4.21 (m, 2×H)

EXAMPLE 8(6-mercaptohexoxyhydroxyphosphinyl)oxy-N,N,N,-trimethylethaniniumhydroxide, inner salt

The compound was prepared by a method analogous to that of Example 6,using 6-bromohexan-1-ol in place of 12-bromododecan-1-ol.

¹ H-NMR (CD₃ OD), 200MH_(z), 1.39 (m, 4×H), 1.66 (m, 4×H), 2.68 (t,2×H), 3.22 (s, 9×H), 3.60 (m, 2×H), 3.85 (q, 2×H).

EXAMPLE 9

The compounds of Examples 6, 7 and 8 were coated onto silver metalsubstrates using the method of Example 5 and were tested using theassays described above, The results were as follows:

    ______________________________________                                     % reduction            % reduction                       % reduction   IgG            fibrinogen activated platelet                                     surface plasmon    Example immunoassay                       immunoassay   resonance    ______________________________________    7       33         33            24    8       65         58            24    6       80         76            83    ______________________________________

We claim:
 1. A compound of formula (I) ##STR17## in which the groups Rare the same or different and each is hydrogen or a straight or branchedC₁ -C₄ alkyl group, n is from 2 to 4, X is a straight or branched C₁₋₃₀alkylene group, or X is a group of formula --(CH₂ CH₂ O)_(b) --, or--(CH₂)_(c) --Ar--(CH₂)_(d) -- where b is from 1 to 20, c and d are thesame or different and each is from 0 to 5, and Ar is a para- ormeta-disubstituted aryl group, which is optionally further substitutedby one or more C₁ -C₄ alkyl groups; and either(a) Y is a valence bond ora divalent functional or heterocyclic group selected from--(X')C(═T)N(H)--, where X' is a straight or branced C₁₋₃₀ alkytenegroup, or a group of formula --(CH₂ CH₂ O)_(b) --, or --(CH₂)_(c)--Ar--(CH₂)_(d) -- where b is from i to 20, c and d are the same ordifferent and each is from 0 to 5, and Ar is a para- ormeta-disubstituted aryl group, which is optionally further substitutedby one or more C₁ -C₄ alkyl groups and T is oxygen or sulphur,--C(═T)--where T is oxygen, sulphur or NH, --C(═T)N(H)-- where T is oxygen,sulphur or NH, and --Het--Y¹ -- where Y¹ is a single bond, oxygen orsulphur, or an alkoxy or alkylthio group containing from 1 to 10 carbonatoms, and Het is a heterocyclic group; and Z is hydrogen or a group--SZ¹ where Z¹ is an alkyl, cycloalkyl, alkylcycloalkyl, aryl,alkylaryl, heterocyclic, alkylheteroyclic group or a group of formula(II): ##STR18## where Y, X, R and n are as hereinbefore defined; or (b)Y is a trivalent alkylene group, Z is a group --SZ¹ and Z¹ is analkylene group, unsubstituted or substituted by alkyl, aryl, alkylaryl,cycloalkyl or alkylcycloalkyl groups and bonded to the group Y so--Y--S--Z¹ form a 5 to 8 membered ring containing a disulphide linkage;or a hydrate thereof.
 2. A compound according to claim 1 in which Z ishydrogen.
 3. A compound according to claim 1 in which X is an alkylenegroup which is a group of formula --(CH₂)_(a) --, in which a is from 1to
 30. 4. A compound according to claim 3 in which a is from 12 to 18.5. A compound according to claim 1 in which Y is a valence bond.
 6. Acompound according to claim 1 in which each of the groups R is methyland n is
 2. 7. An article having a coated metal surface, which comprisesa metal coated on the surface with a compound of formula (I) or ahydrate thereof as claimed in any one of claims 1 to
 6. 8. An articleaccording to claim 7 in which the metal surface is a silver or goldsurface.
 9. A process for useing a compound of formula (I) or a hydratethereof as claimed in any one of claims 1 to 6, wherein a metal surfaceis rendered biocompatible, said process comprising applying to the metalsurface a coating of a compound of formula (I) or a hydrate thereof asclaimed in any one of claims 1 to
 6. 10. A process according to claim 9in which the metal surface is a silver or gold surface.
 11. A compoundof formula (VI), (VII) or (X) ##STR19## where Q is halogen or aprotected thiol group, Y is a valence bond or a divalent heterocyclicgroup and X, R, and n are as defined in claim 1; or ##STR20## where Q¹is halogen or a readily displaceable leaving group, T is oxygen orsulphur, and X, R and n are as defined in claim 1; or ##STR21## where Q¹is halogen or a readily displaceable leaving group, T is oxygen orsulphur, and X, R and n are as defined in claim
 1. 12. A compoundaccording to claim 11, which is a compound of formula (VI), in which Qis a protected thiol group which is a thioether or thioester group.